R68G impairs surface Fas expression and binding to FasL. The BW5147.3 (BW) cell line was transduced with lentiviruses encoding WT or mutated Fas, along with GFP as a marker for transduction. GFP⁺ cells were sorted and used for experiments. (A) Cell surface Fas expression was analyzed with flow cytometry using an anti-Fas antibody, SM1/23. Similar results were obtained with DX2 (data not shown). (B) FasL binding was analyzed with flow cytometry using recombinant human FasL. (C) Intracellular trafficking of Fas was analyzed with immunoblotting. Where indicated, samples were treated with endoglycosidase H (EndoH). PLC-γ1 was used as a loading control. (D) Apoptosis was analyzed by incubating BW cells with recombinant human FasL or cycloheximide for 6 hours and staining with annexin V. (E) Dominant negativity was analyzed by transducing WT-transduced BW cells with empty vector, WT or mutated Fas. Fas-mediated apoptosis was measured as in panel D. All experiments were performed 3 times. Means and standard errors of the mean are shown. **P ≤ .01, ****P ≤ .0001, 1-way analysis of variance. n.s., P > .05.